chr13-32370401-G-T

Position:

• chr13-32370401-G-T

Variant summary

Our verdict is Pathogenic. Variant got 12 ACMG points: 12P and 0B. PVS1_Moderate PM2 PP5_Very_Strong

The NM_000059.4(BRCA2):​c.8332-1G>T variant causes a splice acceptor, intron change involving the alteration of a conserved nucleotide. The variant was absent in control chromosomes in GnomAD project. In-silico tool predicts a pathogenic outcome for this variant. 3/3 splice prediction tools predicting alterations to normal splicing. Variant has been reported in ClinVar as Likely pathogenic (★★).

• Frequency: Genomes: not found (cov: 32)
• Consequence: BRCA2 NM_000059.4 splice_acceptor, intron
• Scores: Splicing: ADA: 1.000
• Clinical Significance: Pathogenic/Likely pathogenic criteria provided, multiple submitters, no conflicts P:7
• Conservation: PhyloP100: 9.74

Genes affected

BRCA2 (HGNC:1101): (BRCA2 DNA repair associated) Inherited mutations in BRCA1 and this gene, BRCA2, confer increased lifetime risk of developing breast or ovarian cancer. Both BRCA1 and BRCA2 are involved in maintenance of genome stability, specifically the homologous recombination pathway for double-strand DNA repair. The largest exon in both genes is exon 11, which harbors the most important and frequent mutations in breast cancer patients. The BRCA2 gene was found on chromosome 13q12.3 in human. The BRCA2 protein contains several copies of a 70 aa motif called the BRC motif, and these motifs mediate binding to the RAD51 recombinase which functions in DNA repair. BRCA2 is considered a tumor suppressor gene, as tumors with BRCA2 mutations generally exhibit loss of heterozygosity (LOH) of the wild-type allele. [provided by RefSeq, May 2020]

BRCA2 (HGNC:):

ACMG classification

Verdict is Pathogenic. Variant got 12 ACMG points.

• PVS1: Splicing +-2 bp (donor or acceptor) variant, product NOT destroyed by NMD, known LOF gene, truncates exone, which is 0.015111631 fraction of the gene. Cryptic splice site detected, with MaxEntScore 5.1, offset of 14, new splice context is: gtccatatttctgctaacAGtac. Cryptic site results in frameshift change. If cryptic site found is not functional and variant results in exon loss, it results in inframe change.
• PM2: Very rare variant in population databases, with high coverage
• PP5: Variant 13-32370401-G-T is Pathogenic according to our data. Variant chr13-32370401-G-T is described in ClinVar as [Likely_pathogenic]. Clinvar id is 185300.Status of the report is criteria_provided_multiple_submitters_no_conflicts, 2 stars. Variant chr13-32370401-G-T is described in Lovd as [Pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	Protein	UniProt
BRCA2	NM_000059.4	c.8332-1G>T		splice_acceptor_variant, intron_variant		ENST00000380152.8	NP_000050.3

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Protein	Appris	UniProt
BRCA2	ENST00000380152.8	c.8332-1G>T		splice_acceptor_variant, intron_variant		5	NM_000059.4	ENSP00000369497.3
BRCA2	ENST00000530893.7	c.7963-1G>T		splice_acceptor_variant, intron_variant		1		ENSP00000499438.2
BRCA2	ENST00000614259.2	n.*390-1G>T		splice_acceptor_variant, intron_variant		2		ENSP00000506251.1

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome Cov.: 31

GnomAD4 genome Cov.: 32

ClinVar

Significance: Pathogenic/Likely pathogenic

Submissions summary: Pathogenic:7

Revision: criteria provided, multiple submitters, no conflicts

Submissions by phenotype

Breast-ovarian cancer, familial, susceptibility to, 2 Pathogenic:2

Pathogenic, criteria provided, single submitter	clinical testing	Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA), c/o University of Cambridge	Oct 02, 2015	- -
Pathogenic, criteria provided, single submitter	clinical testing	All of Us Research Program, National Institutes of Health	Jul 11, 2023	The c.8332-1G>T variant in the BRCA2 gene is located at the canonical splice site of intron 18 and is predicted to inflict acceptor loss (SpliceAI delta score: 0.99), resulting in alternative splicing and disrupted protein product. The variant has been reported in 3 unrelated individuals with breast/ pancreatic cancer (PMID: 29297111, 35171259, 22729890). Experimental analysis of cDNA showed that this variant abolishes the natural 3? splice site of exon 19 and leads to the activation of a cryptic splice site 14 bp downstream. The mutated transcript shows a 14-bp deletion at the beginning of exon 19. This change is predicted to generate a truncated protein (p.Ile2778Tyrfs*15) (PMID: 21735045). Loss-of-function variants of BRCA2 are known to be pathogenic (PMID: 8988179, 11897832, 29446198). The variant is reported in ClinVar (ID: 185300). The variant is absent in the general population database (gnomAD). Therefore, the c.8332-1G>T variant of BRCA2 has been classified as pathogenic. -

Hereditary cancer-predisposing syndrome Pathogenic:2

Likely pathogenic, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Aug 16, 2023	This variant causes a G to T nucleotide substitution at the -1 position of intron 18 of the BRCA2 gene. Splice site prediction tools predict that this variant may have a significant impact on RNA splicing. Although functional studies have not been reported for this variant, it is expected to result in an absent or non-functional protein product. A functional RNA study reported that a variant impacting the same splice site, c.8332-1G>C, causes a 14 nucleotide deletion in exon 19 and is expected to result in a frameshift and premature translation stop signal (PMID: 21735045). This variant has been reported in at least two individuals affected with breast cancer and an individual affected with pancreatic cancer (PMID: 22729890, 30199306, 33471991, 35171259). This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). Loss of BRCA2 function is a known mechanism of disease (clinicalgenome.org). Based on the available evidence, this variant is classified as Likely Pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Ambry Genetics	Mar 19, 2020	The c.8332-1G>T intronic pathogenic mutation results from a G to T substitution one nucleotide upstream from coding exon 18 of the BRCA2 gene. This nucleotide position is highly conserved in available vertebrate species. This alteration was observed in an individuals with a personal and family history of early onset breast cancer (Becker AA et al. Breast Cancer Res. Treat., 2012 Aug;135:167-75; Abulkhair O et al. J Glob Oncol, 2018 08;4:1-9). In silico splice site analysis predicts that this alteration will weaken the native splice acceptor site and will result in the creation or strengthening of a novel splice acceptor site. RNA studies have demonstrated that this alteration results in abnormal splicing in the set of samples tested (Ambry internal data). Another alteration impacting the same donor/acceptor site (c.8332-1G>A) has been shown to have a similar impact on splicing (Menéndez M et al. Breast Cancer Res Treat, 2012 Apr;132:979-92). Alterations that disrupt the canonical splice site are expected to cause aberrant splicing, resulting in an abnormal protein or a transcript that is subject to nonsense-mediated mRNA decay. As such, this alteration is interpreted as a disease-causing mutation. -

Hereditary breast ovarian cancer syndrome Pathogenic:2

Likely pathogenic, criteria provided, single submitter	clinical testing	Women's Health and Genetics/Laboratory Corporation of America, LabCorp	May 26, 2017	Variant summary: The BRCA2 c.8332-1G>T variant involves the alteration of a conserved intronic nucleotide. One in silico tool predicts a damaging outcome for this variant. 4/5 splice prediction tools predict a weakening of the canonical splice donor site while 3/5 splice prediction tools predict the variant to activate a cryptic splice donor site. However, these predictions have yet to be confirmed by functional studies. The variant of interest has not been found in a large, broad control population, ExAC in 121260 control chromosomes. This variant was reported in one study in a BC patient with extensive BC history (Becker_Breast Cancer Res Treat_2012). In addition, multiple clinical diagnostic laboratories/reputable databases classified this variant as pathogenic. Taken together, this variant is classified as likely pathogenic. -
Likely pathogenic, criteria provided, single submitter	clinical testing	Labcorp Genetics (formerly Invitae), Labcorp	Feb 18, 2022	In summary, the currently available evidence indicates that the variant is pathogenic, but additional data are needed to prove that conclusively. Therefore, this variant has been classified as Likely Pathogenic. Studies have shown that disruption of this splice site results in deletion of 14 nucleotides of exon 19 and introduces a premature termination codon (PMID: 21735045). The resulting mRNA is expected to undergo nonsense-mediated decay. ClinVar contains an entry for this variant (Variation ID: 185300). Disruption of this splice site has been observed in individual(s) with clinical features of BRCA2-related conditions (PMID: 22729890, 29446198, 30199306). This variant is not present in population databases (gnomAD no frequency). This sequence change affects an acceptor splice site in intron 18 of the BRCA2 gene. RNA analysis indicates that disruption of this splice site induces altered splicing and may result in an absent or disrupted protein product. -

not provided Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

GeneDx

Feb 01, 2022

Canonical splice site variant expected to result in aberrant splicing, leading to the in-frame deletion of exon 19, which is located in the critical DSS1 contacting residue of the DNA binding domain (Yang 2002); Not observed at significant frequency in large population cohorts (gnomAD); In silico analysis supports a deleterious effect on splicing; Also known as 8560-1G>T; Observed in individuals with a personal or family history of breast cancer in published literature (Becker 2012, Abulkhair 2018); This variant is associated with the following publications: (PMID: 30199306, 22729890, 29446198) -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_addAF	Pathogenic	0.43	D
BayesDel_noAF	Pathogenic	0.38
CADD	Pathogenic	36
DANN	Uncertain	1.0
Eigen	Pathogenic	1.2
Eigen_PC	Pathogenic	1.1
FATHMM_MKL	Pathogenic	1.0	D
GERP RS		5.3

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.94
SpliceAI score (max)		0.99		Details are displayed if max score is > 0.2
DS_AG_spliceai		0.92		Position offset: 15
DS_AL_spliceai		0.99		Position offset: 1

Find out detailed SpliceAI scores and Pangolin per-transcript scores at spliceailookup.broadinstitute.org

Publications

LitVar

Other links and lift over

dbSNP: rs397507979; hg19: chr13-32944538;